Production of glutamic acid



United States Patent 3,201,323 PRODUCTION OF GLUTAMEC ACID John D.Douros, Jn, West Chester, and Andr R. Brillaud and Robert W. Eltz,Media, Pa., assignors to Sun Oil Company, Philadelphia, Pa, acorporation of New Jersey No Drawing. Original application Sept. 13,1963, Ser. No. 308,637. Divided and this application Sept. 25, 1964,Ser. No. 399,357

9 Claims. (Cl. 195-4) This application is a division of our copendingapplication Serial No. 308,637, filed September 13, 1963, now abandoned.

This invention relates to the oxidation of hydrocarbons. Moreparticularly, this invention relates to the fermentation of hydrocarbonsto produce L-glutamic acid. Still more particularly, it is concernedwith the conversion of hydorcarbons to L-glutamic acid in the freestate, and to the substantial exclusion of D-glutamic acid by thefermentation of hydrocarbons with a certain strain of glutamicacid-producing microorganism.

The preparation of L-glutamic acid by fermentation processes is wellknown in the prior art. In each of these processes, however, there hasbeen employed as the sole source of carbon and energy a carbohydrate orother complex oxygenated organic compound. Such a source of energy in afermentation process is disadvantageous because substrates of this sort,and particularly carbohydrates, are relatively expensive raw materials.

Therefore, it is an object of this invention to provide a process forthe production of L-glutamic acid utilizing an inexpensive substratewhich can readily be converted to L-glutarnic acid in high yield. Afurther object is to produce L-glutamic acid without the concurrentformation of D-glutamic acid, thus avoiding the problems of isolationand separation of L-glutamic acid from a racemic mixture.

It has now been found, in accordance with the present invention, thatthese objects can be achieved by growing the hereinafter specifiedmicroorganism on inexpensive hydrocarbon substrates as essentially thesole carbon source to yield L-glutamic acid. When such a fermentation iscarried out under the conditions and with the microorganism described indetail below, L-glutamic acid is produced in high yield substantiallyfree of D- glutamic acid. That hydrocarbon substrates can be employed inthis manner is particularly surprising since most work with hydrocarbonsheretofore has resulted in the formation of carbon dioxide and water asthe principal fermentation products rather than commercially valuableoxygenated organic compounds.

The microorganism employed in accordance with this invention belongs tothe genus Nocardia and the species globerula but cannot be identified asbeing the same as any strain described in Bergeys Manual ofDeterminative Bacterialogy, 7th Edition. This microorganism therefore isconsidered to be a new strain, and a culture of this new strain has beendeposited in the American Type Culture Collection in Washington, DC, andis designated as Nocardia gioberula ATCC 15076. This microorganism, ashas now been discovered, has an outstanding ability to converthydrocarbons to L-glutamic acid in high yield. While various othermicroorganisms can utilize hydrocarbons as their source of carbon and"ice to some extent produce L-glutamic acid in the fermentation, nonehas been found that is comparable to this new strain Nocardia globerulawith respect to the production of this particular amino acid.

Nocardia globerula ATCC 15076 possesses the following culturalcharacteristics:

(1) Gram staingram variable, long rods to cocci,

chains (2) Methylene blue staingranules, long rods to cocci (3) Acidfast stainnot acid fast, rods to cocci (4) Kligler iron agargood growth,moist, raised cream colored (5) Methyl redVoges Proskauerboth negative(6) Gelatin liquefactiongood growth, no liquefaction (7) Nitratebroth-nitrite negative (8) Colony descriptionnutrient agar-creamcolored,

smooth, raised, moist (9) Litmus milksedirnent, no noticeable change(10) Loefflers blood serumgood growth, flesh colored,

raised, smooth, moist (11) Dorset egg mediumluxurious growth, smooth,

moist, orange (12) Sims mediumno H S, Indole negative (13) Glycerolagar-good growth, small cream colored colonies (14) Starch agar-goodgrowth, white, smooth, raised,

doesnt hydrolyze starch 7 (l5) Potato dextrose agarscant growth, raisedtan,

moist (l6) Carbohydrate test in phenol red broth using 0.5% specificcarbohydrate substrate (a) Mannitol-no acid, no gas (b) Levulose-noacid, no gas (c) Lactose-no acid, no gas (d) Inositolno acid, no gas (e)Saccharose-no acid, no gas (f) Arabinoseno acid, no gas (g) Maltose-noacid, no gas (h) Dextrose-no acid, no gas The nutrient medium employedfor the culture of the aforesaid species of this invention to produce L-glutamic acid can vary considerably, but should contain, in addition tothe hydrocarbon substrate as essentially the sole source of carbon, asource of nitrogen and salts. The type of hydrocarbon substrate employedcan vary widely ranging from lower alkyl compounds such as methane orethane to substituted aromatics such as dimethylnaphthalenes. Where thehydrocarbons are volatile, it is preferred to use a closed system toinsure maximum utilization of the substrate by the microorganism and toprevent loss of the volatile substrate into the atmosphere. Otherspecific hydrocarbons which have been found to be useful in this processare such compounds as tetrahydronaphthalene, dodecylbenzene, decane,n-hexadecane, and the like. Mixtures of these and other hydrocarbons arelikewise suitable. Thus, generally speaking, any refinery cuts whichcontain large amounts of normal parafiins in the C -C range, such asBahia gas oil or fuel oil No. 2 can be employed as substrates in thisinvention.

The quantity of hydrocarbon substrate employed in this process may rangefrom 1 to 8% of the total nutrient medium and preferably should be from3 to 6%. It is desirable in carrying out this process that thehydrocarbon substrate be introduced into the fermentation broth in smallincrements so that the hydrocarbon is present in the nutrient medium inamounts below growthlimiting concentrations. Thus, for example, wheresmall amounts of hydrocarbon are being employed, it has been foundpreferable to introduce at the beginning of the fermentation, as littleas 0.25% of the total hydrocarbon to be added, then, after twelve hours,an additional 0.10%, while after twenty-four hours an additional 1% maybe introduced into the fermentation broth. Depending upon the totalamount of hydrocarbon to be utilized, proportionately larger amounts maybe added at twelve to twenty-four hour periods thereafter until thefermentation is complete.

Examples of suitable nitrogen sources are urea, soybean meal, ammoniumsalts such as ammonium sulfate, ammonium phosphate and the like. Othernitrogen sources can be used but are somewhat less effective. Care mustbe exercised in maintaining a suitable pH when ammonia or ammonium saltsare used as nitrogen sources. Mixtures of two or more of these materialscan also be used. The concentration of suitable nitrogen source materialin the nutrient medium is desirably from about 0.5 to 2% The characterof the mineral salts used in the nutrient medium can vary to some extentbut in any event the nutrient medium should contain substantial amountsof phosphorus and magnesium salts in addition to a nitrogen source asmentioned above. Additionally certain trace mineral salts should bepresent, and a suitable mineral salts composition for this purpose is asfollows:

Compound: ,ug./ liter FeSO CuSO -5H O 197 H BO 57 MnSO H O 3 l ZnSO -H O193 Na MOO4' CoCl -6H O 100 5 CaCl 1000 In addition to the foregoingconstituents of the nutrient medium, a source of growth stimulatingmaterials can be used at a concentration of from about 0.01 to 0.1%.Material such as distillers solubles, yeast extract and other substancesof this nature well known in the fermentation field are especiallyeffective.

The process is desirably carried out at a pH of from about 6.0 to 8.0and preferably at a pH of from 7.4 to 8.0. As the culture grows andglutamic acid is produced, the medium tends to become more acidicTherefore, it may be necessary to adjust the pH of the fermentationmedium with a base such as ammonium hydroxide periodically.Alternatively, buffering materials can be used, in which case they canbe conveniently added to the nutrient medium prior to fermentation 'bydiluting the ingredients with an aqueous solution of alkali metalhydrogen phosphates to obtain the desired volume of fermentation broth.One such suitable buffering solution having a pH of 7.4 can be preparedby mixing an aqueous solution of 11.88 grams of Na I-IPO -2H O per literof water and a solution of 9.08 grams of KH PO per liter of water, andmixing them in a 4:1 ratio.

The culturing of the microorganisms in accordance with the process ofthis invention is carried out under aerobic conditions. While sufiicientaeration is generally obtained in small flasks by mechanical agitationduring the fermentation period, it is important in larger scaleoperations that the broth be stirred mechanically and that sterile airbe introduced into the vessels by known methods. It has been generallyfound desirable to introduce from about one-half to one volume of airper minute for each volume of fermentation broth. For optimum yields ofL-glutamic acid, fermentation with the microorganism employed inaccordance with this invention is conducted at about 34 C., andpreferably at about 30 C. for from 48 to 96 hours. Typical yields ofL-glutamic acid after about 72 to 96 hours of incubation of the organismNocardia gl0berula ATCC 15076 under the above conditions are 14 gramsper liter.

The microorganism inoculum is desirably prepared by introducing a 2 ml.wash from a culture slant into 100ml. of a complex carbohydrate nutrientmedium contained in a 500 ml. flask where it is grown for 16 to 24hours. A 5 to 10% inoculum is then obtained from this culture broth byintroducing 5 to 10 parts of this broth into 100 parts of mineralsalts-hydrocarbon fermentation medium described above.

The L-glutamic acid produced in accordance with this process can beconveniently recovered from the fermentation broth by several methodsknown in the fermentation art; as, for example, by first separating thecells and other solid matter by filtration or centrifugation, andthereafter acidifying the fermentation liquor with a mineral acid suchas hydrochloric acid, followed by adsorption of the L- glutamic acid ona weakly basic anion exchange resin such as Rohrn & Haas CompanysAmberlite IR-4B. The amino acid is then removed from this resin byelution with dilute hydrochloric acid, and concentration of the eluateto a small volume. L-glutamic acid hydrochloride can then be obtainedfrom this solution by known crystallization methods.

Alternatively, since the microorganism itself will contain a highproportion of glutarnic acid in combined form, the whole brothcontaining the microorganism first can be treated With an equal volumeof a strong mineral acid such as hydrochloric acid. This acidified brothis then heated for several hours at about 100 C., preferably underpressure, and the released L-glutamic acid is recovered by passage ofthe filtered broth over a column of acid-washed alumina. The alumina canthen be eluted with a suitable solvent such as a dilute alkali to obtainthe L-glutamic acid in purified form following concentration andcrystallization of the eluate.

The amount of L-glutamic acid in a particular fermentation broth can bedetermined not only by actual isolation of the amino acid in the mannerdescribed above, but also by chemical and bio-assay methods. These assaymethods are set forth in detail below:

1. Enzymatic method.The procedure employed is based on that described inAgricultural and Food Chemistry, v01. 5, No. 6, pp. 448457 (1957),wherein dried dead cells of E. coli suspended in a solution buffered atpH 5.0 are used as a reagent. This suspension contains enzymes, one ofwhich quantitatively decarboxylates L-glutamic acid to 'y-amino-butyricacid, and another of which hydrolyzes L-glutamic acid followed bydecarboxylation. The enzyme liberates one mole of carbon dioxide fromone mole of L-glutamic acid. Pyridoxal phosphate functions as thecoenzyme of L-glutamic acid decar'boxylase. From 1.5 to 2.5 mg. ofL-glutamic acid can be measured manometrically in the Warburg apparatuswith a precision within 1% in accordance with this procedure.

2. Chemical methods-This method is based on the procedure described inU.S. Patent No. 3,032,474 wherein culture filtrates containing glutarnicacid are spotted on Whatrnan No. 1 filter paper, gauging the volumeapplied so as to contain 10 to 90 mg. glutamic acid. A standardglutarnic acid solution is spotted on the same sheet of paper at levelsof 10, 30, 50, and mg. to enable constitution of a calibration curve.Unknowns and standard glutarnic acid solutions are run in duplicates ondifferent sheets of paper. The papers are allowed to develop for 20 to24 hours by the descending method. Phenol saturated with an aqueoussolution containing 6.3% sodium citrate and 3.7% monobasic sodiumphosphate is used as the developing solvent. It is found to give a goodseparation of glutamic acid spots from other ninhydrin reacting spotspresent in the filtrates.

Following overnight drying in a current of air, the chromatograms aresprayed lightly with a solution of 0.1% ninhydrin in water-saturatedn-butanol. The glutamic acid spots are localized at R values of about0.23 after heating the papers at 80100 C. for about two minutes.

Quantitation of the glutamate in the spots is performed by the method ofSmith and Agiza, Analyst, 76,623 (1951). Each glutamic acid spot is thencut into small pieces and placed in 1 ml. of water in a graduated testtube. Two ml. of citrate buifer solutions (pH 5) and 2 ml. of ninhydrinreagent are added in sequence to each tube. The tubes are placed inboiling water and 1 ml. of freshly prepared 0.2% SnCl solution (incitrate buffer of pH 5) is added to each tube. After heating for 15minutes, the tubes are removed and cooled in the dark for 10 minutes.The liquid in each tube is made up to 10 ml. by addition of a saturatedNaCl solution. The color containing complex is then extracted from theaqueous phase by shaking with 5 ml. of n-butanol. The clear butanolsolution is pipetted into a Klett tube and the intensity of the purplecolor is measured by a Klett- Summerson photoelectrocolorimeter, using agreen filter. After correction for the reading of a blank prepared fromthe chromatogram but containing no glutamic acid, a straight linerelationship is found to exist between color intensity and concentrationof glutamic acid over a range of 10 to 90 mg. The glutamic acid contentof the unknown filtrates is calculated from their respective standardcalibration curves.

The following examples are specific illustrations of the invention:

Examples A series of four fermentations was carried out in 500 ml.sterilized dispo-plugged Erlenmeyer flasks, using a sterile nutrientmedium of the following composition:

Compound: Concentration MgSO mg./liter 400 (NI-I SO mg./liter 3000 (Na)HPO mg./liter 3000 FeSO '7H O g/liter" 1000 CuSO 5H O ,ug./liter 197 HBO ug/liter" 57 MnSO H O ,ug./liter 31 ZnSO 'H O g/liter" 193 Na MoO21-1 g/liter- 35 CoCl '6I-l O .tg./liter 100 NiCl -6H O g/liter" CaClg/liter" 1000 The pH of the above medium was adjusted to 7.5 with NH OH,and 100 ml. of this medium were introduced into each flask. The mediumwas inoculated with 3% vegetative inoculum of Nocardia globerula ATCC15076, which had been grown on decane for 36 hours, and the fermentationwas conducted on a rotary mechanical shaker at 300 r.p.m. at 30 C. for72 hours. The fermentation broth in each flask was periodicallyadjusted, as necessary, with additional amounts of concentrated NH OH inorder to maintain the pH at 7.5. To each flask 8.8 mg. of decanesubstrate were added initially; 1760 mg. of decane were added after 24hours and the same amount was add- Flask: acid (gm/liter) 1 1.1 2 2.5 31.8 4 2.0

The invention claimed is:

1. A process for the production of L-glutamic acid which comprisessubjecting a hydrocarbon to the action of Nocardia globerula ATCC 15076under aerobic conditions, and recovering the resulting L-glutamic acid.

2. A process for the production of L-glutamic acid from hydrocarbonswhich comprises cultivating under aerobic conditions Nocardia globerulaATCC 15076 in a nutrient medium comprising a source of nitrogen and ahydrocarbon as the essential source of carbon, and recovering theresulting L-glutamic acid from the fermentation broth.

3. The method of synthesizing L-glutamic acid from hydrocarbons whichcomprises dispersing a hydrocarbon in an aqueous nutrient mediuminoculated with Nocardia globerula ATCC 15076, subjecting the inoculatedmixture to incubating conditions for a sustained period of time, andrecovering L-glutamic acid from the resulting fermentation broth.

4. A process according to claim 1 wherein said conditions include a pHin the range of 6.0-8.0 and a temperature in the range of 2534 C.

5. A process according to claim 4 wherein said hydrocarbon is a normalparaflin of the C C range.

6. A process according to claim 2 wherein said conditions include a pHin the range of 6.08.0 and a temperature in the range of 2534 C.

7. A process according to claim 6 wherein said hydrocarbon is a normalparaffin of the C -C range.

8. Method according to claim 3 wherein said conditions include a pH inthe range of 6.0-8.0 and a temperature in the range of 2534 C.

9. Method according to claim 8 wherein said hydrocarbon is a normalparaffin of the C -C range.

References Cited by the Examiner UNITED STATES PATENTS 3,057,784 10/62Davis et a1 -28 FOREIGN PATENTS 588,846 12/59 Canada.

OTHER REFERENCES Yamada et al., Agricultural and Biological Chemistry,26, No. 9, p. 636, September 1962.

Stewart et al., Journal of Bacteriology, 78, 726430, 1959.

A. LOUIS MONACELL, Primary Examiner.

1. A PROCESS FOR THE PRODUCTION OF L-GLUTAMIC ACID WHICH COMPRISESSUBJECTING A HYDROCARBON TO THE ACTION OF NOCARDIA GLOBERULA ATCC 15076UNDER AEROBIC CONDITIONS, AND RECOVERING THE RESULTING L-GLUTAMIC ACID.